Functional Analysis of Viable Circulating Tumor Cells from Triple-Negative Breast Cancer Patients Using TetherChip Technology

Metastasis, rather than the growth of the primary tumor, accounts for approximately 90% of breast cancer patient deaths. Microtentacles (McTNs) formation represents an important mechanism of metastasis. Triple-negative breast cancer (TNBC) is the most aggressive subtype with limited targeted therapies. The present study aimed to isolate viable circulating tumor cells (CTCs) and functionally analyze them in response to drug treatment. CTCs from 20 TNBC patients were isolated and maintained in culture for 5 days. Biomarker expression was identified by immunofluorescence staining and VyCap analysis. Vinorelbine-induced apoptosis was evaluated based on the detection of M30-positive cells. Our findings revealed that the CTC absolute number significantly increased using TetherChips analysis compared to the number of CTCs in patients’ cytospins (p = 0.006) providing enough tumor cells for drug evaluation. Vinorelbine treatment (1 h) on live CTCs led to a significant induction of apoptosis (p = 0.010). It also caused a significant reduction in Detyrosinated α-tubulin (GLU), programmed death ligand (PD-L1)-expressing CTCs (p < 0.001), and disruption of McTNs. In conclusion, this pilot study offers a useful protocol using TetherChip technology for functional analysis and evaluation of drug efficacy in live CTCs, providing important information for targeting metastatic dissemination at a patient-individualized level.


Introduction
The development of new treatments for breast cancer (BC) was achieved by advances in technology; however, metastasis, the primary cause of cancer-related deaths, is still not well understood and is largely incurable. Metastasis is associated with the presence of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in peripheral blood and bone marrow, respectively [1]. The cellular processes involved in metastasis include invading the stroma, avoiding immune surveillance by inhibiting antitumor processes, altering and adapting to the tissue microenvironment, and developing resistance to treatment methods [2]. biomarkers in TNBC, and their presence was linked to patients' outcomes, offering new therapeutic opportunities for this challenging BC subtype [20]. However, there is still a requirement to improve treatment options for people with early stage TNBC in order to minimize the risk of metastasis.
The technical challenges of imaging nonadherent tumor cells are a major obstacle to comprehending how tumor cells react to the nonadherent microenvironments of metastasis, such as the bloodstream or lymphatic system. TetherChip is a microfluidic device that was engineered to prevent cell adhesion with an optically clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization [21]. The thermal imidization process applied to the TetherChip nanosurface on commercially available microfluidic slides enables an impressive capture rate of up to 98% of tumor cells using lipid tethers [21]. Notably, the application of time-lapse microscopy reveals that the distinctive McTNs present on nonadherent tumor cells are rapidly destroyed during chemical fixation. However, when these McTNs are tethered to the TetherChip surface, their structural integrity is effectively preserved both after fixation and post-isolation from blood samples [21]. Remarkably, TetherChips exhibit exceptional stability for over 6 months, facilitating their transportation to remote locations. This microfluidic device presents a pioneering platform that goes beyond enumeration, aiming to enable functional phenotype testing in CTCs. The ultimate objective is to identify patient-specific, antimetastatic therapies [21]. In the present study, we use this novel cell tethering technique, recently developed by Professor S.S Martin's lab at the University of Maryland [21], to isolate viable CTCs from TNBC patients and performed functional analysis for the metastatic capacity of CTCs, McTN formation, and expression of important biomarkers such as GLU, VIM, PD-L1, and CTLA-4, following (FDA-approved) vinorelbine treatment. The current study focused on TNBC not only because of the limited options for targeted therapies, but also because of the higher prevalence of McTNs in this BC subtype [7,9], which provided an opportunity to observe and investigate these structures and evaluate the efficacy of drugs targeting them. Therefore, the aim of this study in using the TetherChip technology was to evaluate the efficacy of drugs on viable CTCs within a microenvironment that simulates the nonadherent conditions of the bloodstream and to investigate the metastatic potential of these cells through the formation of McTNs. By replicating bloodstream conditions, we aimed to gain valuable insights into the behavior of CTCs. Moreover, the anchorage of free-floating CTCs in the TetherChip platform allowed the visualization of microtentacles, which cannot be observed in adherent conditions. This offers a deeper understanding of the mechanism of McTN expansion and the impact of distinct drugs on these structures. Therefore, our protocol using TetherChip technology offers valuable contributions to the understanding of CTC biology and therapeutic approaches at a personalized level.

Patient's Samples and Cytospins' Preparation
The following inclusion criteria were applied for patients' selection: chemotherapynaïve patients (n = 20) with histologically documented triple-negative breast cancer (TNBC), aged > 18 years old, with either metastatic (n = 7) or early stage (n = 13) disease. We chose patients before the initiation of any treatment cycle to avoid drug-induced alterations in the CTCs' phenotypes. Written informed consent was obtained from all participants, and the study received approval from both the ethics and scientific committees of our institution (15/12/21-6734). To collect peripheral blood samples, 10 mL of blood was drawn from patients' veins using EDTA as an anticoagulant. The initial 5 mL of blood was discarded to prevent contamination from skin epithelial cells during the sampling procedure. Ficoll-Hypaque density gradient was used to isolate peripheral blood mononuclear cells (PBMCs). Blood was centrifuged at 1800 rpm for 30 min at 4 • C. The blood components were separated based on their densities. Red blood cells (RBCs) and other heavy components settled at the bottom of the tube, while PBMCs remained suspended, creating a characteristic ring that was carefully isolated by pipetting under sterilized conditions. Cells were then washed twice with PBS and centrifuged at 1500 rpm for 10 min. Aliquots of 500,000 cells were centrifuged at 2000 rpm for 2 min on glass slides [22,23]. Cytospins were dried up and stored at −80 • C, as we have previously reported [23][24][25][26].

Cell Cultures
Breast cancer cell lines MDA-MB-231 and MDA-MB-436 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in high-glucose Dulbecco's modified eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; PAN-Biotech, Passau, Germany) and 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA). A patient-derived colon CTC-MCC-41 cell line was obtained under a collaboration agreement between the University Hospital of Montpellier and the University of Patras and cultured in a Roswell Park Memorial Institute (RPMI; Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 1% L-glutamine, 1% ITS (insulin, transferrin, and selenium; Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS, 20 ng/mL EGF (Thermo Fisher Scientific, Waltham, MA, USA), and 10 ng/mL FGF (Thermo Fisher Scientific, Waltham, MA, USA). Finally, cells (4 million) from TNBC patients were cultured in RPMI medium supplemented with 1% L-glutamine, 1% ITS, 10% FBS, 20 ng/mL EGF, and 10 ng/mL FGF. Cells from TNBC patients were cultured for 4-5 days. This approach aimed to deplete as many PBMCs as possible to reduce blood-cell-related noise in the TetherChip. The cell culture also enriched the CTC population and provided the critical number of cancer cells to study drug efficacy in the TetherChip. The cells were cultured in a humid environment containing 5% carbon dioxide (CO 2 ) and 95% air. For subculturing, a solution of 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA, USA) and 5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA) was used. After two hours, all the media including MTT solution (5 mg/mL) were removed. The residual formazan crystals were dissolved in isopropanol, and the absorbance was measured at 555 nm using a 96-well microplate reader (SynergyTM HT, Bio-Tek Instruments, Inc., Winooski, VT, USA).

TetherChip Analysis
For immunofluorescence in TetherChips, 50,000 cells were allowed to tether for 30 min onto TetherChip, then fixed with 3.7% formaldehyde/PBS, washed, permeabilized in 0.1% Triton-X 100/PBS, blocked in 5% FBS/PBS, and incubated overnight at 4 • C. The antibody sequence was the same as in the cytospins. For McTN visualization, wheat germ agglutinin (WGA, Alexa Fluor 488 conjugate, Invitrogen, 1:100), and Hoechst 33258 (1:5000) were diluted in 1% FBS/PBS, added to each channel, and incubated for 1 h at room temperature. Based on the findings of Thompson et al. [12], vinorelbine was used in the present study to treat TNBC patients' CTCs and their McTNs. Following 30 min for the tethering of cells, vinorelbine was added for 1 h onto TetherChip. M30 CytoDeath monoclonal antibody (Sigma Chemical Co., St. Louis, MO, USA) was used to detect apoptosis.

Statistical Analysis
Paired t-test analysis was used to compare the average number of CTCs detected in each patient's cytospins, obtained after Ficoll density isolation, and the number of CTCs of the same patient located in TetherChips following 4-5 days of culture, the average number of CTCs before and after vinorelbine treatment in TetherChips, and the viability of cancer cells (MTT). Spearman analysis was used to correlate the different phenotypes in cytospins and TetherChips. A Mann-Whitney test and χ 2 tests were used to compare the expression of PD-L1, CTLA-4, GLU, and VIM before and after 1 h vinorelbine treatment. All statistical analyses were conducted using IBM SPSS statistics version 27 software (IBM, Armonk, NY, USA). A significance level of p ≤ 0.05 was employed to determine statistically significant findings.

Cytospin vs. TetherChip Analysis of TNBC Patients' CTCs
Consequently, we compared the number of CTCs detected in cytospins after Ficoll density isolation (500,000 PBMCs per patient) with the number of CTCs in TetherChips after 4-5 days of culture (50,000 cultured PBMCs were added to TetherChips per patient). The comparison revealed a statistically significant increased number of CTCs (p = 0.006) located in TetherChips ( Figure 2A and Table 1). Moreover, an evaluation of the CTCs' number in TetherChips vs. cytospins among early and metastatic TNBC patients also revealed a significantly higher number of CTCs counted in TetherChips in both settings ( Figure 2B). Consequently, the followed protocol with TetherChip analysis provides dra-matic enrichment of CTCs per patient. The number of isolated CTCs per patient is shown in Table 1.   Early Early Early Early Early
Representative images depicting the expression of all the different biomarkers in CTCs of TNBC patients located in TetherChips are illustrated in Figure 6.  In agreement with the aforementioned results from the BC cell lines, the effect of vinorelbine in the CTC-41 cell line (cultured in nonadherent conditions) revealed that cell viability (77%) remained high but statistically different compared to control cells after 1 h vinorelbine treatment, whereas 24 h vinorelbine treatment induced significant loss of viability of CTC-41 (58%) cells (p < 0.001, Figure 7C).

Effect of Vinorelbine on PD-L1, CTLA-4, GLU, VIM Expression, and Induction of Apoptosis in MDA-MB-436 in TetherChips
To standardize the protocol for studying drug efficacy on tumor cells, using TetherChip technology, we treat the cancer cell line MDA-MB-436 with vinorelbine in a TetherChip. We used this cancer cell line for control experiments because of its superior visualization of McTNs.
Regarding the expression of the immune checkpoint molecules in MDA-MB-436, our findings revealed that the percentage of PD-L1 + cells before and after 1 h vinorelbine treatment was 81% and 54%, respectively ( Figure S1). On the other hand, the percentage of CTLA-4 + cells before and after 1 h vinorelbine treatment was 95% and 77%, respectively.
Regarding the expression of EMT-related molecules (GLU and VIM), the percentage of GLU + cells before and after vinorelbine treatment was 88% and 64%, respectively. Additionally, the percentage of VIM + cells before and after vinorelbine treatment was 88% and 92%, respectively ( Figure S1). Finally, the study of apoptosis on these cells showed that M30 + cells before and after vinorelbine treatment was 5% and 37%, respectively, whereas the same percentages for M30 − cells were 95% and 63%, respectively.
Representative images depicting the expression of all the different biomarkers in MDA-MB-436 cells are illustrated in Figure S2.

Effect of Vinorelbine on PD-L1, CTLA-4, GLU, VIM Expression, and Induction of Apoptosis in TNBC Patients
Initially, to investigate the effect of vinorelbine on the total number of CTCs, we conducted a comparative analysis of the mean number of CTCs prior to and after vinorelbine administration. Our findings showed that there was a significant difference in the average number of CTCs before and after vinorelbine treatment (p = 0.008, Table 2).
Considering the established association between PD-L1 and CTLA-4 with disease progression, as supported by evidence from our published research [20], we performed an investigation of the expression of these immune checkpoint molecules in live CTCs isolated from the TNBC patients. Additionally, we assessed the impact of a 1 h vinorelbine treatment on the expression levels of these molecules. Our findings revealed that the percentage of PD-L1 + CTCs before and after vinorelbine treatment was 92% vs. 77%, respectively (p < 0.001), while the same percentages for PD-L1 − CTCs were 8% vs. 23% (p < 0.001), respectively, indicating a significant effect of vinorelbine treatment on PD-L1expressing CTCs (Figure 8). Considering CTLA-4 expression, the percentage of CTLA-4 + CTCs before and after vinorelbine treatment was 41% and 43%, respectively. Similarly, the same percentages for CTLA-4 − CTCs were 59% and 57%, respectively. These findings indicated no significant effect of vinorelbine treatment on CTLA-4 expression (Figure 8).  Extending our analysis, we explored the effect of vinorelbine on EMT-related molecules (GLU and VIM). We found that the percentage of GLU + CTCs before and after vinorelbine treatment was 89% and 67% (p < 0.001), respectively, whereas the same percentages for GLU − CTCs were 11% and 33% (p < 0.001), respectively, demonstrating that vinorelbine treatment causes a significant reduction in GLU expression (Figure 8). Additionally, the percentage of VIM + CTCs before and after vinorelbine treatment was 98% and 89%, respectively, whereas the same percentages for VIM − CTCs were 2% and 11%, respectively; however, this finding did not reach a statistical significance (Figure 8).
Upon establishing the impact of vinorelbine on the expression levels of PD-L1, CTLA-4, GLU, and VIM, we proceeded to investigate whether vinorelbine could elicit apoptotic responses in the live CTCs of TNBC patients. We found that the percentage of M30 + CTCs before vs. after 1 h vinorelbine treatment was 16% and 30% (p < 0.010), respectively, whereas the same percentages for M30 − CTCs were 84% and 70% (p < 0.043), respectively (Figure 8). These findings provide compelling evidence that exposure to vinorelbine is capable of inducing apoptotic responses in the CTCs of TNBC patients.

Vinorelbine Effects on McTNs
Furthermore, we determined the effect of 1 h vinorelbine treatment on McTNs of TNBC patients' CTCs. Our experiments revealed an impressive disruption of these structures in CTCs after treatment (Figure 9).

Discussion
It is widely accepted, as recently published in Nature Reviews Clinical Oncology [13], that there is a great need to find treatments that can reduce metastasis and to move away from over-reliance on response evaluation criteria for solid tumors (RECIST), which are based primarily on the imaging measurement of tumor growth rather than metastatic features. Furthermore, results from a clinical trial in 2017 indicated that CTCs can be detected in the bloodstream an average of 6 months before metastasis is observed on a PET/CT scan [28]. Metastatic breast cancer is still an incurable disease, and metastasis is the main cause of death in these patients. Recent improvements in liquid biopsy techniques have demonstrated the prognostic value of CTCs, indicating the importance of CTCs analysis for clinical practice [29]. Findings from a recent study, using TN human cancer cell lines (MDA-MB-231 and MDA-MB-436), showed that the microtubule-depolymerizing drug, vinorelbine, reduced the metastatic phenotypes of MCTNs, reattachment, and tumor cell clustering rather than tumor cell viability [12]. These findings also suggested that vinorelbine may be a more effective treatment for TNBC than previously thought, as it appears to reduce metastasis more than primary tumor growth [12].
Taking these into account in the current study, we focused on patients with TNBC because it is the most aggressive phenotype, it rapidly progresses to metastasis, and it is characterized by a limitation of targeted therapies. We evaluated the metastatic capacity of live CTCs from TNBC patients under conditions similar to the bloodstream microenvironment (nonadherent) using the TetherChip device. More specifically, we isolated live CTCs and characterized them in relation to their metastatic capacity, McTN formation, and expression of potentially important biomarkers such as GLU, VIM, PDL-1, and CTLA-4, following (FDA-approved) vinorelbine treatment.
Although the most aggressive subset of CTCs that can survive in the bloodstream and form metastasis are largely unknown, it has been shown that metastatic BC cell lines adopt a morphologic cellular reattachment phenotype, which is associated with the presence of McTNs [9,11,30]. A higher McTN number is found in more invasive BC cell lines [9]. Furthermore, the molecular mechanisms that are linked to McTNs are correlated with a greater risk of metastasis and a poorer prognosis for the patient [8].  Figure 3). This finding agrees with our former studies showing that McTNs on CTCs isolated from BC patients appeared to play a role in facilitating communication between CTCs, as well as interactions between CTCs and surrounding blood cells [1].
To gain a better comprehension of the biology of CTCs, especially the metastasisinitiator CTCs, functional assays are urgently needed. Unfortunately, the current CTC capture methods have low yield, making it difficult to obtain enough viable CTCs for these types of functional assays. To address this issue, it is necessary to increase the number of CTCs and develop more efficient CTC capture methods [31]. Technologies like CTC-Chip, CellSearch, and other similar systems are designed to capture CTCs based on specific phenotypes or surface markers expressed on tumor cells [32,33]. These systems use antibodies or antibody-coated surfaces to target and capture CTCs with specific characteristics, such as the expression of epithelial cell adhesion molecule (EpCAM) or other epithelial markers. On the other hand, the TetherChip technology allows the capture and observation of CTCs without relying on specific markers. It aims to study the metastatic potential of cancer cells by facilitating the visualization of McTNs. Therefore, TetherChip utilizes a lipid-based tethering approach to immobilize CTCs, enabling their examination and analysis [21]. This technology has the potential to capture and visualize different phenotypic subpopulations of CTCs without being limited to specific surface markers. It also provides a more unbiased approach to study drug efficacy on CTCs' subpopulations. In our study, we cultured PBMCs, isolated by Ficoll-Hypaque density gradient, for 4-5 days and subsequently analyzed them in TetherChips. The comparison between the number of CTCs found in each patient's sample directly after blood collection (cytospins) and the number of CTCs of the same patients analyzed in TetherChips showed that there was a significantly increased number of CTCs in the TetherChip device, suggesting that this protocol could improve the step of CTC enrichment. Subsequently, a functional assay can be applied to get a profile of viable CTCs and their response to treatment.
Moreover, we compared the phenotypic characteristics of patients' CTCs in cytospins and TetherChips. Our results revealed that, in cytospins, the expression levels of PD-L1, CTLA-4, and GLU were notably increased in the metastatic setting of TNBC patients ( Figure 4). This finding is in line with the results of our recent research [20]. Regarding the comparison between TetherChips and cytospins, there was no statistically significant differences regarding different phenotypes, implying that our protocol does not influence the physiology of CTCs. The only phenotype that was increased in TetherChips was the GLU-positive one. Particularly, GLU expression in CTCs was significantly increased (p = 0.01) in TetherChips compared to in cytospin preparations in both early and total TNBC patients ( Figure 5). This could be attributed to the fact that GLU participates in McTN formation and aids cancer cell migration and invasion by providing structural support [34].
Additionally, the augmented stability of microtubules supported by GLU may contribute to the resistance of cancer cells to chemotherapy [34].
In this study, we also aimed to investigate the effect of vinorelbine treatment on the viability of CTCs based on previous findings on human cancer cell lines [12]. Therefore, the TNBC cell lines (MDA-MB-231 and MDA-MB-436), as well as the patient-derived colon CTC-MCC-41 line, were treated with vinorelbine for 1 h and 24 h. Our findings revealed a substantial reduction in cell viability across all three cancer cell lines, underscoring the profound impact of vinorelbine treatment on their viability (Figure 7).
Consequently, we assessed the effect of vinorelbine on patients' CTCs (in TetherChips) and on their distinct phenotypes that we have recently shown to be relevant to patients' outcomes, such as PD-L1, CTLA-4, GLU, and VIM [20] (Figure 8). The presented findings demonstrate a noteworthy reduction in PD-L1 and GLU expression following vinorelbine treatment (p < 0.001). High levels of PD-L1 expression have been associated with worse OS of early TNBC and NSCLC patients and with shorter PFS of metastatic breast cancer (MBC) patients [35][36][37][38][39]. On the other hand, high GLU expression has been linked to significantly decreased OS and PFS of NSCLC and BC patients, respectively. Its overexpression has also been shown in McTNs, associated with EMT pathways [1,35]. Based on our data, vinorelbine treatment decreases the expression of these two biomarkers (PD-L1 and GLU) in CTCs, implying that vinorelbine could be an effective treatment by inhibiting metastasis and improve patient outcomes.
One additional question was whether vinorelbine treatment could induce apoptosis in CTCs. Our results indicated a significant increase in the percentage of M30 + CTCs following vinorelbine treatment, suggesting that vinorelbine induced apoptosis in CTCs of TNBC patients, reinforcing the importance of this drug for targeting metastatic spread ( Figure 8). Furthermore, our results have shown that vinorelbine disturbs McTNs in patients' CTCs ( Figure 9) after one hour of treatment, in agreement with previous studies, showing that vinorelbine reduces the metastatic phenotypes of McTNs in MDA-MB-231 cells [12]. There are different strategies to disrupt McTNs such as kinesin inhibitors or curcumin treatment [3,40]. Furthermore, ionomycin and thapsigargin cause a sudden rise in cytoplasmic Ca 2+ levels, which suppress the McTNs in the MDA-MB-231 and MDA-MB-436 metastatic breast cancer cell lines [41].
The small number of patients included in this study and the exclusive focus on TNBC patients is a limitation of this study. Expanding the sample size and including other cancer types would provide a more comprehensive understanding of microtentacles and drug response across different malignancies. Furthermore, the investigation of more cytokines and adhesion molecules in relation to McTNs and drug efficacy could unravel the complex interactions in the bloodstream. Furthermore, the in vitro experimental approach does not fully replicate the complexity of the in vivo tumor microenvironment. Future research incorporating in vivo models would help bridge this gap and validate the clinical relevance of our findings. However, besides these limitations, to the best of our knowledge, this is the first study testing drugs to inhibit metastatic potential of CTCs by reducing microtentacle formation using live CTCs from TNBC patients.

Conclusions
Our study highlights the importance of the functional analysis of CTCs using Tether-Chips technology as a valuable tool for real-time investigation of drug efficacy on isolated, cultured CTCs. Our results also revealed that vinorelbine can elicit rapid changes in critical biomarkers such as PD-L1 and GLU. It also induced the apoptosis of patients' CTCs and disturbed the cytoskeletal organization of McTNs. Funding: This research has been co-financed by the European Regional Development Fund of the European Union and Greek funds through the Operational Program Competitiveness Entrepreneurship and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T1EDK-01562). This research was also funded by a grant from the Research Committee of the University of Patras via the "C. CARATHEODORI" program (project code: 81351), and by R01-CA154624 from the National Cancer Institute (USA). C.A.-P. is supported by la Fondation ARC pour la Recherche sur le cancer and les Fonds de dotation AFER pour la recherche médicale.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics and scientific committees of our institution (15/12/21-6734).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data presented in this study are available upon request from the corresponding authors.